Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.
Abbreviations used: ROS, reactive oxygen species; GPX, glutathione-dependent peroxidase; TcGPX, T. cruzi GPX; cGPX, cytosolic GPX; PHGPX, phospholipid hydroperoxide GPX; Ni-NTA, Ni2+-nitrilotriacetic acid; BiP, homologue of immunoglobulin heavy-chain binding protein; TcCPX, T. cruzi cytosolic peroxiredoxin; TcMPX, T. cruzi mitochondrial peroxiredoxin; ER, endoplasmic reticulum; CHEFE, contour-clamped homogeneous electric field electrophoresis.
The nucleotide sequence for TcGPXII reported in this paper has been deposited in the EMBL/GenBank Nucleotide Sequence Database under accession number AJ313313.